Human IFN beta Assay Kit

Type I interferons, interferon alpha (IFN-α) and interferon beta (IFN-β), play essential roles in the innate immune response to viral infections. IFN-β is produced by fibroblasts, macrophages, and dendritic cells, as well as endothelial cells. The expression of the IFN-β gene is controlled by transcription factors like IRF-3, c-Jun/ATF-2, and NF-kappaB downstream of Pattern Recognition Receptors, such as Toll-like receptors. In particular, IFN-β is the primary functional readout of cGas-cGAMP-STING pathway activation. Once secreted, IFN-β binds and activates the IFNAR1/IFNAR2 receptor complex, which relies on STAT1/2 transcription factors. Phosphorylated STAT1/2 leads to the induction of anti-viral gene expression.

Cisbio's Human IFN-β kit is a homogeneous add and read assay to measure IFN-β secretion in supernatant. It offers a fast, efficient alternative to ELISA.

Sample size 14 µL
Final assay volume 20 µL
Kit components Lyophilized standard, frozen detection antibodies, activation reagent, buffers & protocol
LOD & LOQ (in Diluent) 7 pg/mL & 19 pg/mL
Range 19 – 4,000 pg/mL
Time to result 3h @ RT
Calibration NIBSC (00/572) value (IU/ml) = 0.44 x HTRF hIFNβ value (pg/ml)
Species Human only
Download Insider tips for PBMC isolation Technical Note
Download cytokine release from fresh blood samples application notes

Assay principle

The assay protocol, using a Cisbio 96-well low volume or a 384-well small volume white plate (20 µL final), is described below. Cell supernatant, sample, or standard are dispensed directly into the assay plate for detection by HTRF® reagents. The antibodies labeled with the HTRF donor and acceptor are pre-mixed and added in a single dispensing step, to further streamline the assay procedure. The assay can be run in 96 to 1536-well plates by simply resizing each addition volume proportionally.

The 4 Parameter Logistic (4PL) curve is commonly recommended for fitting an ELISA standard curve. This regression enables the accurate measurement of an unknown sample across a wider range of concentrations than linear analysis, making it ideally suited to the analysis of biological systems like cytokine releases.

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Cytokines assay principle add

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Cytokines assay principle read

(1) Assay data analysis:

  • The "Guidelines for data reduction" button will show you how to deal with HTRF data processing and 4PL 1/y2 fitting.
  • The "MyAssays" button will direct you to a free online software able to run human IFNbeta analysis.

 

How to use HTRF cytokine assays

How to measure multiple cytokines from 1 sample

Analytical performance

Precision

Intra assay (n=24)

Sample Mean [IFN-β] (pg/mL) CV
1 33 4%
2 321 3%
3 3900 5%
  Mean CV 4%

 

Inter assay (n=4)

Sample [IFN-β] (pg/mL) Mean (delta ratio) CV
1 62 374 10%
2 329 1628 7%
3 1739 5690 9%
    Mean CV 9%

 

Assay validation

1. STING pathway activation by cGAMP triggers hIFNβ release in PBMC

400,000 PBMC were seeded in a 96 well plate under 100µL of cell culture medium, then stimulated with 50µL of cGAMP used at 67µM. After an overnight incubation, 14µl of supernatant were transferred into a white detection plate and human IFNβ was quantified by HTRF.

2. LPS efficiently induces hIFNβ release in THP1 cells.

400,000 THP1cells were seeded in a 96 well plate under 100µL, then stimulated with 50µL of LPS used at 20µg/ml. After 4h30 incubation, 14µl of supernatant were transferred into a white detection plate, and human IFNβ was quantified by HTRF.

3. Recombinant THP1 cells expressing constitutively activated STING variant spontaneously releases hIFNβ.

400,000 of THP1-WT cells (wild type) and THP1-S154 cells (THP1-Dual™ KI-hSTING-S154 Cells, Invivogen) were seeded in a 96 well plate, under 100µL of cell culture medium. After 4h30 of stimulation, 14µl of supernatant were transferred into a white detection plate, and human IFNβ was quantified by HTRF.

4. dsDNA mimetic (CDS agonist) induces hIFNβ secretion

400,000 THP1-S154 cells (THP1-Dual™ KI-hSTING-S154 Cells, Invivogen) were seeded in a 96 well plate under 100µL of cell culture medium, then treated with dsDNA mimetic (CDS Agonist – LyoVec, Invivogen). After an overnight incubation, 14µl of supernatant were transferred into a white detection plate, and human IFNβ was quantified by HTRF.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
Human IFN beta kit - 500 tests62HIFNBPEG
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Human IFN beta kit - 10,000 tests62HIFNBPEH
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Companion products

DescriptionCat. noProduct insertMSDS
Human IFN beta kit - 3 Standards62HIFNBCD3
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Human IFN beta kit - Standard62HIFNBCDA
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